Microdissection-based methods for determining genomic features of single chromosomes

ABSTRACT

The present provides a microdissection-based method for identifying a genomic feature present within a visible chromosome region. The method includes steps of: (a) micro-dissecting a single copy of a chromosome to obtain a visible chromosome region; (b) amplifying the visible chromosome region to obtain amplified single chromosome DNA; and (c) subjecting the amplified single chromosome DNA to micro-array analysis whereby such analysis identifies at least one genomic feature present within the visible chromosome region. The method is applicable to determining genomic features including, but not limited to, genomic DNA size, gene content, DNA breakpoint, or DNA polymorphism (e.g., single nucleotide polymorphisms).

FIELD OF THE INVENTION

This invention relates generally to the identification andcharacterization of genomic features of chromosomes. More specifically,the present invention is directed to microdissection-based methods thatfacilitate direct determination of genomic features of either normal orabnormal single chromosomes.

BACKGROUND OF THE INVENTION

Chromosome aberration is a hallmark of cancer, which provides valuableclues to pinpoint the candidates for cancer-related genes.Cancer-specific chromosome aberrations often result in alterations inthe structure and/or the dosage of cancer-causing genes [Balmain, et al.Nat. Genet. 33 Suppl: 238-244 (2003)]. Examples include Bcr/Abl fusiongene caused by the translocation between chromosomes 9 and 22(Philadelphia chromosome) in chronic myeloid leukemia (CML), and loss ofa copy of the Rb gene caused by the deletion of the proximal region ofthe long arm of chromosome 13 in retinoblastoma. Chromosomerearrangement in cancers may also result in alterations in other genes,which may not be the primary cause of cancer, but have an impact oncancer susceptibility and/or cancer progression, and thus have a greatvalue in cancer risk assessment, diagnosis and prognosis. Therefore,genes and their alterations involved in all clonal and recurrentchromosome abnormalities should be carefully characterized.

Most cancer chromosome abnormalities are detected through cancercytogenetics studies. Once a clonal chromosome structure abnormality isdetected, researchers may further define the genomic region(s) involvedin the abnormality by FISH or other DNA hybridization-based techniquesto “walk through” the abnormality with various cloned genomic sequences.The gene content in the defined region(s) can be disclosed by matchingthe genes that have been mapped in the regions from the human genomedatabases. This approach can narrow down the candidates forcancer-related genes, from which the actual cancer-causing andcancer-risk genes may be identified by genotype-phenotype correlationand functional studies. Such a strategy is referred to ascytogenetics-based positional cloning and is so far the most successfulstrategy for identifying cancer-related genes, particularly fromhematological cancers. However, this strategy has limitations.

First, it is heavily relied on in standard chromosome studies and FISHto define the genomic regions that are involved in cancer chromosomeaberrations. Due to limited resolution and often poor cancer chromosomemorphology, determining the genomic origins of cancer chromosomeabnormalities can be particularly difficult. It is often furthercomplicated when the aberrations are only detected in a small number ofcancer cells that are mixed with a large population of cells with anapparently normal karyotype. Because of this limitation, the genomicorigins of many observed cancer chromosome abnormalities cannot beidentified for further analysis.

Second, the cytogenetics-based positional cloning is not astraightforward, high-throughput strategy; the analytical process ofthis strategy is cumbersome and labor-intensive. It may take severalmonths for a skillful researcher to fully analyze one abnormality todetermine the genomic regions, breakpoints and gene content involved inthe abnormality. In addition, such an analysis usually requires asignificant amount of cancer specimens, but that are not alwaysavailable. Therefore, only a very small fraction of observed cancerchromosome abnormalities have been thoroughly characterized forpositional cloning. Linkage analysis can also be used to revealcandidate loci for positional cloning. However, this method is morecommonly used for studying constitutional Mendelian diseases and rarefamilial cancer cases; its application seems to be limited in geneticanalysis of sporadic cancers, perhaps due to the complexity of thecancer genetics and genomics.

Genome-wide screening for genomic DNA copy number imbalance is anotherstrategy for cancer genetic analysis, including restriction landmarkgenome scanning [Hayashizaki, et al., Electrophoresis 14: 251-258m(1993)], comparative genomic hybridization (CGH) [Kallioniemi, et al.Science 258: 818-821 (1992)], high-throughput quantitative PCR[Ginzinger, et al. Cancer Res. 60: 5405-5409 (2000)] and molecularsubtraction techniques, such as representational display analysis[Lisitsyn, et al. Science 259: 946-951 (1993)]. These global screeningtechniques, particularly the array-based CGH [Albertson, et al., Nat.Genet. 25: 144-146 (2000)], are powerful tools to detect genome-wide DNAsequence dosage change, including deletion and duplication, in thecancer genome. However, these techniques also have limitations. Theyusually cannot detect balanced chromosomal rearrangements that oftenresult in cancer-causing gene fusion and/or breaking apart, such as theBcr/Abl fusion gene in CML and the MLL gene split in various types ofleukemia. In addition, the capability of these techniques to detect DNAcopy number imbalance can be complicated or impaired in mixed cellpopulations. Unfortunately, most cancer specimens are contaminated withmore or less normal cells, and genomic changes in most cancers areheterogeneous; cancer tissues are often mixed with multiple cell lineswith multiple clonal and/or non-clonal (random) genomic abnormalities.Furthermore, these techniques may not directly reveal the dosage ofindividual genes involved in the unbalanced genomic regions. Forexample, the resolution of the current array CGH is about a hundredthousand base pairs of genomic 5 DNA [Albertson, et al., Nat. Genet. 25:144-146 (2000); Vissers, et al., Am. J: Hum. Genet. 73: 1261-1270(2003).], which is better than that of the cytogenetic analysis, but isnot enough to determine the dosage alterations in individual genes.Array-based gene expression analysis is also a powerful tool for cancerstudies, which reveals expression patterns of all known or predictedgenes in cancers [Hanash, S. Nat. Rev. Cancer 4: 638-644 (2004)]. Theexpression level of each gene can be measured and genes with similarexpression levels or with similar functions can be grouped for furtheranalysis. Since many genes may show a similar expression level and theabnormal expression may or may not represent the primary genetic changein cancer, the array expression analysis is an excellent tool for cancerbiology studies rather than identification of cancer-causing genes andprimary genetic changes. From a technical point of view, the best way tocharacterize a chromosome rearrangement is directly analyzing thegenomic DNA from the abnormal chromosome, which increases the efficiencyand accuracy of detecting the genomic content, gene content and possiblemutations involved in the regions. As of yet, however, most currenttechnical strategies do not have the ability to directly characterizedetectable chromosome aberrations.

In addition, none of the techniques described above can directly uncoverthe genomic features, such as DNA sequence variations, of the abnormalchromosome regions. Such information is an important part of molecularprofile of cancer, which will facilitate cancer epidemiology studies,risk assessment, diagnosis and prognosis. Genomic sequence variation orpolymorphism is an important feature of the genome. The most common typeof variation is single nucleotide polymorphism (SNP) that is defined asa single nucleotide variation at a locus with the frequency of the minorallele greater than 1% in at least one population [Risch, N. J. Nature405: 847-856 (2000).]. It is estimated that the human genome containsmore than 15 million SNPs [Botstein, et al. Nat. Genet. 33 Suppl:228-237 (2003)]. These polymorphisms are valuable markers for geneticassociation studies, because they are frequently linked withdisease-related genes or traits. It is apparent that SNPs are notinherited randomly in the same chromosome; instead, they are ofteninherited as phased combinations of specific alleles in particularpopulations. Therefore, analyzing phased SNPs or SNP haplotypes providesa more informative approach to study genetic associations.

In general, haplotype is a combination of linked polymorphic alleles ona single chromosome. A given homologous chromosome pair in the diploidgenome has two haplotypes, representing maternal and paternal origins[The International HapMap Consortium. Nature 426, 789-796 (2003)].Haplotypes of the human genome appear to be organized as discrete blockswith an average size of 9-18 kb in length (ranging from less than 1 kbto more than 170 kb) due to linkage disequilibrium (LD) [Gabriel, S. B.et al. Science 296, 2225-2229 (2002)]. Linked polymorphic alleles withineach block tend to act as a single multi-site allele with limitedhaplotype diversity [Wall, J. D. & Pritchard J. K. Nat. Rev. Genet. 4,587-597 (2003)]. The haplotype blocks represent the evolution,inheritance and recombination histories of the genome. Thus, analyzinghaplotype blocks of highly condensed polymorphic markers, such as SNPs,provides a powerful tool for genetic association studies [Bostein, D. &Risch, N. Nat. Genet. Suppl. 33, 228-237 (2003); Crawford, D. C. et al.Am. J. Hum. Genet. 74, 610-622 (2004); Drysdale, C. M. et al. Proc.Natl. Acad. Sci. USA 97, 10483-10488 (2000)]. Uncovering SNP haplotypesof the abnormal chromosome regions in cancer should also be of greathelp for tracing the origins of the abnormal alleles, following-up theprogression of the abnormalities, identifying low-penetrancecancer-related traits, and studying drug response and prognosis.

However, unambiguously determining haplotypes of SNP blocks, especiallylarge blocks, is particularly challenging due to technical limitations.There have been two broad categories of tools for unambiguoushaplotyping: genotyping family pedigrees and directly genotyping SNPs ona single chromosome of interest [Crawford, D. C. & Nickerson, D. A.Annu. Rev. Med. 56, 303-320 (2005)]. The former is expensive,time-consuming and requires DNA samples from several generations, whichare not always available. In addition, accurately assigning SNP phaseusing family-based methods becomes increasingly difficult as more lociare considered. Meanwhile, the latter currently relies on enrichment ofDNA of single-chromosome origin using complicated methods, such assomatic cell hybrid and multi-step allele-specific PCR, which is alsotime-consuming and expensive. These limitations make it difficult toapply unambiguous SNP haplotype analysis to either individual- orpopulation-based genetic association studies.

Taken together, effectively identifying disease-causing and disease-riskgenes as well as their genomic alterations/variations, particularly fromcancer-associated clonal chromosome aberrations and constitutionalmosaicism from mixed cell populations, remains a great challenge ingenetic research.

SUMMARY OF THE INVENTION

The present invention provides a chromosome microdissection-basedstrategy for detailed molecular analysis of any cytogenetically visiblechromosome regions from a single cell. It is a straightforward “what yousee is what you get” approach that possesses several unique advantages.It allows the direct isolation of any normal or abnormal chromosomes orchromosome regions from targeted single cells without the interferenceof the surrounding cells. The isolated chromosomes or chromosome regionsare efficiently amplified for high-resolution genomic analysis, such asdetermination of the breakpoints, gene content, DNA content and geneticvariations of the target regions. This strategy provides a unique toolto analyze clonal acquired abnormalities in cancer cells and mosaicconstitutional abnormalities observed in a variety of genetic disorders.In addition, this strategy is particularly useful for unambiguouslydetermining haplotypes of any genomic regions without the requirement offamily pedigrees and genotypes, LD and population genomic information,or complicated statistical analysis and computing. For example, it canquickly determine SNP haplotypes of any single or multiple haplotypeblocks, chromosome regions or chromosomes regardless their genomiclocations and sizes, as well as the frequency of LD in the targetregions. Furthermore, this strategy has a strong potential to be usedfor genome-wide and population-based haplotype analysis when it can beapplied with large scale SNP array technologies. Amplified singlechromosome DNA samples may be analyzed using SNP arrays or,alternatively, subjected to genotyping by primer extension and DNAsequencing. For the genome-wide analysis, one may separate and amplifyhaploid chromosome sets of an individual, and directly determine thehaplotypes of each haploid set using appropriate SNP arrays. This newstrategy may largely facilitate both individual- and population-basedgenetic association studies. In addition, this strategy may also beapplied to haplotype analysis of species other than human.

Accordingly, the present provides in a first embodiment a method ofidentifying a genomic feature present within a visible chromosomeregion. Such a method includes steps of: (a) micro-dissecting a singlecopy of a chromosome to obtain a visible chromosome region; (b)amplifying the visible chromosome region to obtain amplified singlechromosome DNA; and (c) subjecting the amplified single chromosome DNAto micro-array analysis whereby such analysis identifies at least onegenomic feature present within the visible chromosome region.

In certain embodiments, the method includes the additional step ofperforming reverse fluorescence in situ hybridization (FISH) on achromosome preparation using the amplified single chromosome DNA toidentify a genomic origin of the visible chromosome region. With thisinformation available, the micro-array analysis may then be at leastpartially targeted to the genomic origin of the visible chromosomeregion; knowledge of the genomic origin is particularly valuable whereregion specific micro-array analysis is performed.

In a preferred embodiment, the genomic feature identified by the methodis a single nucleotide polymorphism (SNP). If a plurality of singlenucleotide polymorphisms (SNPs) are identified, then a SNP haplotype ofthe visible chromosome region is provided by the method. As well, wherethe method is repeated using a corresponding haploid chromosomehomologue, then SNP haplotypes of a chromosome homologue set areprovided by the method.

In certain embodiments, the visible chromosome region comprises avisible aberration. The chromosome micro-dissected in the method ispreferably obtained from a single cell, most preferably isolated from apopulation of heterogeneous cells of human origin. The chromosome regioncarrying an aberration can be amplified and characterized byhigh-resolution genomic DNA arrays for determining the breakpoints, genecontent and DNA content which are involved in the aberration. While thecharacterization of abnormal chromosomes is a preferred application ofthe present invention, it should be understood that the invention isequally applicable to the characterization of genomic features in normalchromosomes of human or non-human origin.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Evaluation of microdissection and amplification of single-copy21q and determining of SNP alleles on single 21qs. (a) Evaluation byFISH. Fluorescence-labeled DNA amplified from a single 21q hybridizesexclusively back to the 21qs with strong and even hybridization signals(arrows). (b) Evaluation by locus-specific PCR. The gel photographydemonstrates all 14 tested alleles that are amplified from a single 21q.(c) Testing of SNP alleles at each locus by primer extension asdescribed in Example 1. Arrows indicate the allele peaks. The homozygouslocus rs2836015 shows only an identical single allele peak in each 21qhomologue and in the genomic DNA; while the heterozygous locus rs2836019shows a different single peak in each homologue and both peaks in thegenomic DNA. (d) Determining of SNP alleles by DNA sequencing. Eachamplified allele is also sequenced using ABI 3700 DNA analytical system.The arrows indicate the corresponding SNPs in the same samples tested in(c). H-homologue sample; G-genomic DNA.

FIG. 2. SNP haplotypes detected in five individuals. The dash lineindicates the haplotype block B000966 that was predicted having limitedhaplotype diversity across different ethnic groups [Patil et al. Science294:1719-1723 (2001)]. Two haplotypes of this block are shared byunrelated individuals (GCACT shared by individuals 1 and 2 and CCGTTshared by individuals 3 and 4). Individual 4 and 5 are mother and son,who share a same haplotype (individual 4-H2 and individual 5-H1),demonstrating that the inheritance of a specific haplotype betweengenerations can also be readily analyzed by the present invention.

FIG. 3. Several different haplotypes of Haplotype Block B000966determined in a group of 5 individuals, including 5 reported and 2unreported haplotypes.

FIG. 4. FISH and micro-array analysis of aberrant human chromosome 1 intwo unrelated pediatric patients.

DETAILED DESCRIPTION OF THE INVENTION I. In General

Before the present materials methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, cell lines, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”,“characterized by” and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, cell lines,vectors, animals, instruments, statistical analysis and methodologieswhich are reported in the publications which might be used in connectionwith the invention. All references cited in this specification are to betaken as indicative of the level of skill in the art. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); and Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C. C. Blackwell, eds., 1986).

II. The Invention

The present invention provides a strategy that can readily andreliability characterize genomic features contained within a visiblechromosome region of a single chromosome. The invention is particularlywell-suited in certain embodiments to characterize visible chromosomeaberrations present in, for example, cancer cells. In the abnormalchromosome context, the present technology facilitates theidentification of genes and their genomic changes and/or polymorphisms(e.g., haplotype blocks) associated with chromosomal aberrations. Inother embodiments, the present technology is equally suited tocharacterize normal chromosomes, including the identification of SNPalleles for haplotyping a specific chromosome or, alternatively, anentire genome.

Visible chromosome regions, no matter whether their genomic origins areidentified or not, are characterized according to the invention by: 1)isolating chromosome regions using chromosome microdissection; 2)amplifyng the regions from single copies of chromosomes; and 3)subjecting the amplified single chromosome DNA to high-resolutiongenomic/gene arrays in order to determine a genomic feature including,but not limited to, DNA polymorphisms, genomic intervals, breakpointlocations and the gene content. In more preferred embodiments, methodsaccording to the invention identify SNP alleles present within thevisible chromosome region thusly providing the direct determination of asingle chromosome's haplotype.

In terms of analyzing abnormal chromosomes, particularly thoseassociated with cancer cells, this strategy has unique advantages thatwill overcome current technical difficulties, and greatly facilitate theeffort of understanding cancer genomics and genetics. First, any visiblechromosome aberration can be quickly and precisely isolate bymicrodissection. Where aberrant morphologies are concerned, the genomicorigin of the aberration can also be quickly and precisely determined byreverse FISH. Limited number of abnormal cells in a mixed cellpopulation and poor cancer chromosome morphology will not affect theanalysis, since as low as only one copy of the abnormal region is neededfor dissection and the genomic origins of the abnormalities will bedetermined on normal chromosomes by FISH using dissected DNA as probes.Second, the abnormal region-specific high-resolution genomic/gene arrayanalysis using the dissected DNA will be much more efficient andaccurate than the traditional positional cloning on determining thegenomic and gene content of abnormal regions. Third, this strategyallows the direct determine of SNP alleles on a single copy of abnormalchromosome, which represent the haplotype of the chromosome. Since theabnormal DNA is isolated, other genomic alterations and variations inthe abnormal region may also be directly analyzed. This feature has aparticular significance on cancer genetic research, which makes itpossible to directly identify molecular characteristics or the molecularbasis (the genomic profile) of the genomic regions that are involved incancer-related abnormalities in each individual cancer patient. Suchmolecular basis and profiles will largely facilitate the identificationof genetic factors that influence the genome stability and theindividuals' risk of developing cancer or their ability of responding tothe cancer therapy. Fourth, this strategy can characterize virtually anytype of chromosome abnormalities from any dividing cell populations,mixed or not mixed, once the abnormalities are detected by cytogenetics.

Distinct from most of the current strategies, this is the first “whatyou can see, is what you can analyze” strategy, which will allow cancerresearchers to maximally utilize the cancer cytogenetic findings foridentifying cancer-related genes, genomic alterations and variations.This straightforward and efficient technical strategy makes it possibleto systematically analyze all detectable cancer abnormalities. Ofcourse, the application of the present invention is applicable togenome-wide genomic analysis for both normal and abnormal cell types(e.g., directly measuring the haplotypes of an entire genome).

The present invention combines techniques including microdissection, DNAamplification and DNA array techniques. Such techniques and theirapplication to the present invention are further described below.

Chromosome microdissection is a unique technique of isolatingchromosomes or chromosome regions from animal and plant cells byphysically removing the regions of interest from the chromosomepreparations using a sharp needle that is controlled by amicromanipulator or a laser bean [e.g., Kao, et al., BioEssays 15:141-146 (1993); Kao, F. T. In Methods of genome analysis in plants(edited by Jauhar, P P), CRC Press, New York, pp 329-343 (1996)].Dissected chromosome regions have been amplified by various PCR-basedtechniques [e.g., see Kao, F. T (1996), ibid] and by MultipleDisplacement Amplification (MDA) for a variety of basic research andclinical applications, including construction of region-specific genomiclibraries for genomic analysis, cloning of disease genes, generation ofregion-specific FISH probes and identification of the genomic origins ofchromosome abnormalities. A unique and powerful feature that makes thistechnique superior than others is its capability of isolating andcloning any cytogenetically visible chromosome regions from as few as asingle metaphase cell from any eukaryotic species.

The amplification of dissected DNA is a key step in the presentinvention. Linker adaptor-based and degenerate oligonucleotide primer(DOP)-based PCR techniques are known in the field and may be adapted, asdescribed herein, for use in the present invention. DNA amplified bysuch techniques has been successfully used for constructing genomiclibraries and generating FISH probes [e.g., see Kao, F. T (1996), ibid].Multiple Displacement Amplification (MDA) is a relatively-new technologyfor genomic DNA amplification using a special DNA polymerase, phi29polymerase, which is isolated from the phage phi-29 strain [Dean, et al,Proc. Natl. Acad. Sci. U.S.A. 99: 5261-5266.16-18; (2002); Hosono, etal., Genome Res. 13: 954-964 (2003); . Barker, et al., Genome Res. 14:901-907 (2004)]. This enzyme can efficiently amplify a trace amount ofDNA templates in 30° C. by continuously replacing one parent DNA chainwith a new synthesized one without changing the reaction temperature.Another unique feature of phi29 DNA polymerase is that it amplifieslarge molecules, up to a few hundred kb, and the amplification appearsto be more complete and more even. While microdissected DNA has beenamplified using the techniques described, it is of particular note thatamplified DNA samples from microdissection have not been shown amendableto micro-array analysis outside of the present disclosure.

In general, DNA array is a technology that spots a large number of DNAsamples in a small surface with a very high density for DNAhybridization analysis [Schena, et al., Science 270: 467-470 (1995)].Using arrays, researchers can examine the full complexity of a genome ina single experiment. Arrays have been applied to studies in geneexpression, genome mapping, SNP discrimination, transcription factoractivity, toxicity, pathogen identification and detection, as well asmany other applications.

In the present invention, at least two different types of arrays findparticular utility; SNP array and region-specific high-resolutiongenomic/gene array. Currently, there are several commercially availableSNP array techniques suitable for use in the present inventionincluding, but not limited to, the Affymatrix human SNP array (GenechipMapping 100K set) that contains over 100,000 SNPs genome-wide[Affymetrix, Inc.,http://www.affymetrix.com/products/arrays/specific/100k.affx].High-resolution genomic/gene arrays useful in the present invention havebeen developed and are commercially available from NimbleGen Systems,Inc [Nimblegen System, Inc. illustrative information available atwww.nimblegen.com/products/index.html.]. Such high-density microarraysare manufactured via a proprietary Maskless Array Synthesizer (MAS)technology. This technology generates arrays with extremely highinter-array reproducibility (up to r²>0.99) and very low intra-arraycoefficient of variation (cv<12%) across the array surface. However, thepresent invention is not limited in the array technology it mayincorporate. For example, large genomic clone-based CGH arrays may beutilized to characterize chromosome breakpoints while oligo-based arraysmay be used to uncover the detailed gene content in the tested regions.

Various aspects of the invention are described in further detail in thefollowing subsections.

III. Examples Example 1 Determining Single Nucleotide Polymorphism (SNP)Haplotypes Using Dissected Single Chromosomes

This example describes the unambiguous determination of SNP haplotypesof a selected chromosome region using a technique according to thepresent invention. The disclosed technique includes the steps of singlechromosome microdissection, universal DNA modification, and directanalysis of amplified single-chromosome DNA by primer extension.

The inventors analyzed the haplotypes of 14 SNP loci across the long armof chromosome 21 (21q) in five normal Caucasians, including threeunrelated individuals, a 5 mother and her son. Briefly, the inventorsisolated 10 single-copy 21qs from each individual in five culturedmetaphase peripheral blood cells by chromosome microdissection andamplified each individual 21q using modified degenerate oligonucleotideprimed PCR (DOP-PCR). The quality of the dissection and amplificationwas evaluated by fluorescence in situ hybridization (FISH), in which,two randomly selected amplified 21qs from each individual werehybridized to normal metaphase cells. All tested samples showed strongand even hybridization signals exclusively on 21q (FIG. 1 a), indicatingthat the dissections were accurate and the single-chromosomeamplifications were efficient.

In order to examine the efficiency and coverage of this strategy forhaplotype analysis at both individual block and whole chromosome armlevels, the inventors selected 14 21q-specific SNP loci from HumanGenome Resource, NCBI (http://www.ncbi.nlm.nih.gov/genome/guide/human)for analysis. These loci span approximately 30 Mb along 21q, includingfive that are located on a 10 kb DNA stretch within a known haplotypeblock and nine that are evenly distributed along the rest regions of 21q(FIG. 2) [Patil, N. et al. Science 294, 1719-1723 (2001)]. The SNPalleles were individually amplified from the DOP-PCR products of each21q using locus-specific primers. On average, about 8.2 (range 6-14)loci were amplified from a single copy of 21q. FIG. 1 b shows an exampleof amplification of all 14 SNP loci from a single 21q. Loss of allelesfrom a single dissected chromosome is anticipated because of thepossibility of random DNA damage during the process. It was demonstratedthat alleles lost from a 21q homologue in one cell could be readilyamplified from the same homologue in a different cell of the sameindividual. The tested alleles were also amplified from the genomic DNAof each individual as controls. The inventors then genotyped each 21qand the corresponding control genomic DNA sample using locus-specificprimer extension and DNA sequencing. As expected, only a single alleleat every locus on single-copy 21qs was detected; whereas, mixedheterozygous alleles were detected at some loci in genomic DNA samples(FIGS. 1 c and 1 d). Two alleles detected at a genomic heterozygouslocus of an individual were always detected separately on twocorresponding 21q homologues (FIGS. 1 c and 1 d). These findingsdemonstrate that the amplified DNA samples from dissected singlechromosomes are also suitable for array analysis, particularly SNPanalysis.

Most importantly, the SNP haplotype of a 21q homologue was automaticallyuncovered once the SNP polymorphic alleles on that homologue weredetermined. In the present study, 3-5 metaphase cells from an individualwere sufficient for a full haplotype analysis of the 14 loci; whenpartial loss of alleles occurs on a single chromosome, the completehaplotype of the chromosome can always be determined by comparing allelephases in a few different cells. Using this strategy, the inventorsunambiguously determined the haplotype of the 14 loci on each 21qhomologue from the five tested individuals (FIG. 2). At least two of thehaplotypes determined for Haplotype Block B000966 were unreportedthereby further demonstrating the present invention's utility in theidentification of genomic features (FIG. 3).

Materials and methods related to this example are as follows:

Microdissection and DOP-PCR

Metaphase chromosomes for microdissection were prepared on 24×50 mmcoverslips as previously described [Kao, F. T. and Yu, J. W. Proc. Natl.Acad. Sci. USA 88, 1844-1848 (1991)]. Single chromosome microdissectionand universal amplification was modified from previously publishedmethods [Meltzer, P. S. et al. Nat. Genet. 1, 24-28 (1992); Jordan, B.et al. Proc. Natl. Acad. Sci. USA 99, 2942-2947 (2002)]. Briefly, asingle 21q was dissected with a sharp glass needle using TransferMan NK2micromanipulator (Eppendorf) attached to the microscope, and transferredto 5 μl collection buffer containing 5 mM NaCl, 2 mM MgCl₂, 4 mM Tris-Cl(pH7.5), 200 μM dNTPs and 1 μM equally mixed four degenerate primers(5′-CCGACTCGAGNNNNNNATGTGG-3′, (SEQ ID NO:71)5′-CCGACTCGAGNNNNNNATCATC-3′, (SEQ ID NO:72)5′-CCGACTCGAGNNNNNNTTGAGG-3′ (SEQ ID NO:73) and5′-CCGACTCGAGNNNNNNGATACA-3′. (SEQ ID NO:74))The dissected 21q was then treated with 1U Topoisomerase I (Promega) at37° C. for 20 min, denatured at 96° C. for 10 min and followed by eightcycles of pre-amplification using 1:8 freshly diluted Sequenase Version2.0 DNA polymerase (USB). Each cycle includes 94° C. 1 min, 30° C. 2min, 37° C. 2 min and addition of 0.2U fresh enzyme. Thepre-amplification product was further amplified by PCR in 50 μl reactionsolution containing 2U Taq DNA polymerase (Eppendorf), 1×PCR buffer with1.5 mM MgCl₂ (Eppendorf), 100 μM dNTPs and 0.2 μM mixed degenerateprimers. The PCR cycles include a hot start at 94° C. for 3 min, 35cycles of 94° C. 1 min, 56° C. 1 min and 72° C. 2 min, and a finalextension at 72° C. for 10 min. The PCR products were examined byelectrophoresis on 1% agarose gel.Fluorescent in Situ Hybridization (FISH)

The DOP-PCR products were labeled with Digoxigenin-11-dUTP (Roche) andhybridized to spreads of human metaphase chromosomes according toNon-Radioactive In Situ Hybridization Application Manual (Roche, see,e.g., www.roche-applied-science.com/PROD_INF/MANUALS/). FISH resultswere analyzed and documented using the proprietary Cytovision systemavailable from Applied Imaging.

Isolation of Genomic DNA

Genomic DNA from each tested individual was isolated from blood cellsusing the Wizard Genomic DNA Purification Kit (Promega).

Amplification of SNP Alleles

SNP alleles at each locus were amplified from the DOP-PCR products andthe genomic DNA samples using locus-specific primers. Occasionally, somesamples showed increased background that can be eliminated by anadditional round of nest PCR using primers that are nested within thecorresponding starting PCR products. Locus-specific starting and nestPCR primers for each locus are listed in Table 1. The starting and nestPCR reactions were carried out using a “step-down” protocol, includingone cycle of 94° C. 3 min, 10 cycles of 94° C. 30 sec, 65° C. 30 secwith a decrease of 1.5° C. per cycle and 72° C. 30 sec, and 30 cycles of94° C. 30 sec, 51° C. 30 sec and 72° C. 30 sec, followed by a finalextension at 72° C. for 10 min; the PCR solution (50 μl for each PCRreaction) contains 1 mM MgCl₂, 100 μM dNTPs, 0.2 μM of each forward andreverse primers, 1×PCR buffer and 2U Taq DNA polymerase (Eppendorf). ThePCR products were examined on 3% agarose gel and treated withExonuclease I and Shrimp Alkaline Phosphatase (ExoSAP-IT, USB) to cleanup unincorporated primers and dNTPs for the following primer extensionand sequencing analyses. TABLE 1 Chromosome 21q SNP locus-specific PCRprimers Starting primer (5′→3′) Nest primer (5′→3′) refSNP ID (SEQ IDNO) (SEQ ID NO) rs2824397 GCCAGGGCATGTTTTATAGG (1)GGAGTCTGCTCTTTACTTTAAGC (29) CCACTGTTTTGGCACTGAGA (2)GGCACTGAGAACGAAGGTAA (30) rs2826399 TCCATTCCACTCAACACACG (3)CGCAGACATATACAGGCCATA (31) CAATGACACCCAAAAATTCG (4) CGTCTGTCGTGCATGTTGA(32) rs2828312 TTGAAAGATATCCACTCTCTTCTTCA (5) CCACTCTCTTCTTCATTCTGGA(33) GGAGAGTATGCTTTACATATCAGGAA (6) CAGGAAACTTCTCTTATGGTTCTTC (34)rs2830585 TGATGGTTGCTGACACTTGG (7) GGGAGCAGCGTACCATTG (35)TGCAGTTTGCCTATCGTCAC (8) CCCTGCTCCCAGAAACAAC (36) rs1153292TGACTTCCAGAGGGATGAGC (9) GGATGAGCTGGCCTCTTTTT (37) TCCAAACACAGCCTAGTCACC(10) ATGTGGAACAATGCCACTGA (38) rs1805127 GCCCTTTCTGACCAAGCTGT (11)TGTGGCAGGAGACAGTTCAG (39) AGAAGCCGAAGAATCCCAGT (12) CGTAGAGGGCCTCCAGCTT(40) rs2226357 ATGAAATGTTTGGTATGTTGACCA (13) CCACATGATTAGCATTTTGTAGC(41) GTCGTACCCAATGTCCGAGT (14) TGTCCGAGTTTATTGGTCCTT (42) rs2836015CAGGAGAAATTTCCATTTTTCAA (15) AAAATATGCCTTGTATTTCATATTCAT (43)AGAATAAAGCCTTCTTCAAATGAG (16) AGAATAAAGCCTTCTTCAAATGAG (44) rs2836016GGCCATTGTTGCAGTTTCTT (17) TTCTTTGTGCTTTTCCTGGAG (45)CCTAGGAAAAAGAAGGAAGAGAGA (18) GCTGTAAATGGCAATTAGATCA (46) rs2836019ACCTGCTTGCTGTGGAATTT (19) TTGGGTTATGTCAACATGCAG (47)AGGCTCCTCTCTGCCTGTCT (20) TCTCTGCCTGTCTTATTCAGCTC (48) rs2836021CCTGGTGTGCTCATTTCAGA (21) GCTGCTTGCTGTTTTCTGC (49)TTTTTCACCTTAAAATACCACCAA (22) CAGTCTATCAAAGCATGTTCAGG (50) rs2837501GGCCTTTTATATTCGACATGGA (23) TCTCACCCACAGAGGCTTTT (51)GCACAGGTTACACGTTGTGTC (24) GATTTCACAGTTCCCTCTGCTT (52) rs2839596TTTTCACTCTAAACTGTTCTGTCCA (25) CACATTTTGGCAGCTGGTG (53)TAGTGGGAGTGGCTTCTTGG (26) GCATGGAGAGCACCTGAATC (54) rs2839320TCCACCTGCCTGTTAGGAAC (27) GGAACCAATTTTAATGATAAACTCAA (55)ATGTGATGTGTCCAGCTCGT (28) CGTCCAGCTCCAGGATGAT (56)Primer Extension Analysis

Approximately 50 ng of a locus-specific PCR product was mixed with 0.1μM extension primer, 50 μM locus-specific ddNTP and dNTP mix (see Table2), 1× extension reaction buffer and 2U Thermo Sequenase DNA polymerase(Amersham) in 20 μl for the primer extension reaction. The reactioncycles included an initial denaturation at 95° C. for 3 min followed by50 cycles of 96° C. 10 sec and 58° C. 1 min. Primer extension productswere analyzed using the WAVE Nucleic Acid Fragment Analysis System(Transgenomic) under a fully denaturing condition according to themanufacturer's instruction. TABLE 2 Chromosome 21q SNP locus-specificsingle base extension (SBE) primers SBE primer (5′→3′) Product refSNP IDAlleles (SEQ ID NO) Nucleotide mix length (bp) rs2824397 G/TGAGCTGTCTTTTGTACTCTGCT (57) ddGTP, dATP, dTTP 23 (G)/29 (T) rs2826399C/T CAGCGTTAATATTGTCCATTTCA (58) ddTTP, dCTP, dGTP 24 (T)/28 (C)rs2828312 A/T GGATCTAAATGGGTGGGTAAAG (59) ddATP, dCTP, dTTP 23 (A)/28(T) rs2830585 C/T GGGCATGAGACTGCAGGAG (60) ddTTP, dCTP, dGTP 20 (T)/23(C) rs1153292 A/C TTCTTGGACAGCTTTTCCAG (61) ddCTP, dATP, dGTP 21 (C)/24(A) rs1805127 C/T CCTCCAGCTTGCCGTCAC (62) ddTTP, dCTP, dGTP 19 (T)/22(C) rs2226357 C/G TGTCCGAGTTTATTGGTCCTTA (63) ddGTP, dATP, dCTP 23(G)/27 (C) rs2836015 A/G GAGAAATGCAGTTTTCTATGATGAA (64) ddATP, dCTP,dGTP, 26 (A)/30 (G) dTTP rs2836016 A/G TTTGTGCTTTTCCTGGAGGT (65) ddGTP,dATP 21 (G)/23 (A) rs2836019 C/T GCCAGTAGAGAGGCTAAGTGTCA (66) ddTTP,dCTP, dGTP 24 (T)/26 (C) rs2836021 A/G AAGCATGTTCAGGTATCCTCTTC (67)ddGTP, dATP, dTTP 24 (G)/26 (A) rs2837501 C/T CACAGAGGCTTTTTGGCA (68)ddCTP, dTTP, dGTP 19 (C)/23 (T) rs2839596 A/G TTTGTTCACTACAAGTCCCTTAAAA(69) ddATP, dCTP, dGTP, 26 (A)/31 (G) dTTP rs2839320 C/TGATAAACTCAAGGATGGCATCT (70) ddTTP, dATP, dCTP, 23 (T)/27 (C) dGTPSequencing

The locus-specific PCR products were labeled with ABI PRISM BigDye(Applied Biosystems) using the locus-specific nest PCR primers (seeTable 1) according to the manufacturer's instruction and sequenced onABI PRISM 3700 sequencer (Applied Biosystems)

Example 2 Characterization of Visible Regions of Normal Chromosome 21

This example illustrates a method according to the invention forhaplotype determination of a human chromosome, specifically, humanchromosome 21 [Hattori, et al., Nature 405: 311-321 (2000)]. Thestrategy involves several technical steps, including microdissection ofchromosome regions, universal amplification of dissected DNA, reverseFISH for identification of the genomic origins of the chromosomeregions, region-specific genomic DNA/gene array and genome-wide SNParray analysis. Single copies of dissected chromosome 21 may be obtainedfrom a transformed normal lymphoblast cell line. A suitable cell line isGM 03657 (Coriell Institute; normal karyotype of 46,XY).

Metaphase cells are spread on clean 25×50 mm coverslips and G-banded formicrodissection, as described in our previous studies [Kao, et al.,Proc. Natl. Acad. Sci. U.S.A. 88: 1844-1848 (1991)]. The chromosome andthe chromosome region to be dissected are identified under themicroscope and subseqently dissected with a sharp glass needle using anEppendorf TransferMan NK2 manipulator attached to the microscope. Thedissected chromosome may be delivered in a collection buffer for theuniversal amplification. In this example, single copies of the wholelong arm of chromosome 21 (21q) and a series of regions of 21q withdifferent sizes are isolated for the sake of illustration.

Since the amplification template is only a single copy of a chromosomeregion, several rounds of amplification are needed to generatesufficient amount of DNA for the following analysis. DOP PCR has beensuccessfully used to amplify a single dissected chromosome region forFISH [Meltzer, et al., Nat. Genet. 1: 24-28 (1992)], and to amplifygenomic DNA for genotyping analysis [Cheung, et al., Proc. Natl. Acad.Sci. U.S.A. 93: 14676-14679 (1996); Cheung, et al., Proc. Natl. Acad.Sci U.S.A. 93: 14676-14679(1996)].

Briefly, a single copy of chromosome 21 is treated with topoisomerase I(Promega) for releasing the tension of the double-helix DNA chains, andthen with 5-8 cycles of initial amplification using T7 polymerase (USB)and a degenerate primer (5′-CCGACTCGAGNNNNNNATGTGG-3′ (SEQ ID NO:75),followed by 25 cycles of the first round PCR with Taq DNA polymerase andthe same primer. The PCR products are then treated with shrimp alkalinephosphatase (USB) to clean up the primers and unmatched DNA templates,and then used as the templates for the second round PCR amplificationwith fresh prepared buffer, enzyme and the primer; if needed the thirdround PCR in similar fashion.

Alternatively, dissected DNA is digested with a restriction enzyme andthe resulting DNA fragments are ligated with a linker adaptor on theirboth ends, which serves as the specific primer binding site, then aspecific PCR can be performed. Since primer-specific PCR is used,dissected chromosome regions can be repeatedly amplified to producelarge amount of DNA with high quality and low background, which can bedirectly used for FISH and for array analysis. The amplified DNA canalso be cloned for construction of sequencing-ready genomic libraries,from which each clone can be directly sequenced. If a chromosome regionof interest is separately digested with two different restrictionenzymes for adding linker adaptors for amplification, the sequencecoverage for this chromosome region may increase significantly. This isperhaps thus far the most efficient way to clone abnormal regions and toproduce large stable and pure genomic clones from the abnormal regionsfor genomic analysis. As an alternative to PCR-based amplifications, MDAmay be utilized to amplify dissected single chromosomes. This methoduses phage phi29 DNA polymerase to amplify DNA at 30° C., which canamplify a large amount of high-molecular weight genomic DNA.

Reverse FISH may then be performed to confirm the genomic origins of theamplified DNA. The amplified DNA is labeled with fluorescence-labelednucleotides by PCR, randomly priming or nick-translation (for MDAproducts) as probes, and hybridized to normal metaphase chromosomespreads following standard protocols. FISH results may be analyzed usingan image analysis program (Cytovision, Applied Imaging); with thegenomic origins of the dissected chromosomes identified by locating thehybridization signals on the specific chromosome regions.

Once the genomic origin of a dissected chromosome fragment isidentified, analysis using high-resolution, region-specific genomic/genearrays is performed to characterize the dissected fragment. For example,a series of chromosome 21 region-specific arrays may be obtained fromNimbleGen Systems, a representative provider of such arrays. Aproprietary hybridization system, including protocols, buffers,hybridization cassettes, hardware, and temperature controlinstrumentation that generate highly reproducible and sensitivehybridizations are commercially available. These protocols have beenoptimized to require minimum amounts of sample, while providing ahybridization volume that can be continually mixed over the time-courseof the incubation. Such arrays are useful to reveal the gene content,genomic tag content and the potential breakpoint positions, if any, ofthe dissected chromosome.

SNP and haplotype analysis is then carried out using arrays currentlyavailable for detecting SNPs, such arrays being divided into two groups:locus-specific SNP arrays and the genome-wide global SNP arrays. Aparticularly well-suited array is the Affymatrix human SNP array(Genechip Mapping 100K set) that is a global SNP array containing over100,000 SNPs genome-wide. Since only a single chromosome is analyzed foreach test, the array of SNP alleles detected in that chromosomerepresents the actual haplotype of the chromosome. Haplotype blocks andthe corresponding haplotype tag SNPs in each analyzed chromosome regioncan be determined by matching detected SNP alleles with the haplotypeblock and SNP maps in HapMap and other databases.

Example 3 Characterization of Lymphoma-Specific Chromosome Abnormalities

Lymphomas are a group of heterogeneous hematological malignancies whichoften show chromosome aberrations. Certain lymphoma-specific chromosomeabnormalities have been identified, such as t(8;14)(q24;q32) andt(11;14)(q13;q32) translocations in Burkitt lymphoma and Mantle celllymphoma, respectively. However, the genomic features of many otherclonal chromosome aberrations that are frequently seen in lymphomas,particularly marker chromosomes, remain unknown. Such aberrations can bereadily characterized by the invention. In this example, the inventorscharacterized two cytogenetically undistinguishable chromosome deletionsin only two cells from two unrelated pediatric patients, respectively,to demonstrate the proof of principle. This example involves severaltechnical steps, including microdissection of chromosome regions,universal amplification of dissected DNA, reverse FISH foridentification of the genomic origins of the chromosome regions, andregion-specific genomic DNA/gene array analysis.

Each visible chromosome region containing a deletion was individuallydissected from a single cell from each patient and was amplified inanalogous fashion to that described in the previous Example section. Theamplified abnormal regions were used as probes in reverse FISH toidentify the corresponding genomic origins which were determined tobe1q42.11-1q42.13 (FIGS. 4 a & b). The deleted regions are nottranslocated to the other part of the genome. In addition, the FISHresults indicate that the deletions are apparently not identical,showing different sizes and positions ((FIG. 4 b, the deletions showedas “gaps” in the FISH picture). Amplified DNA from the dissected regionwas then subjected to high-resolution, chromosome 1q42 region-specificoligonucleotide genomic DNA array analysis. This analysis clearlyrevealed two non-overlapped deletions in different patients, consistentwith the FISH findings: the patient #1 has a more distal 10.6 Mbdeletion between 227.4-238 Mb, which contains 67 known genes, and thepatient #2 has a more proximal 5.3 Mb deletion between 218.3-223.6 Mb,which contains 52 known genes (FIG. 4 c, the deletions are indicated bythe background level of 1000). The resolution of this array analysis canbe as high as 1 kb, and the findings using only a single cell from eachpatient unambiguously define two cytogenetically undistinguishabledifferent deletions and provide detailed information for furtherclinical assessment of the two patients. This is the first example ofapplying single-copy microdissected chromosomes to the high-resolutionDNA array analysis.

Those skilled in the art will recognize, or be able to ascertain usingno more then routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention and encompassed by the following claims.

1. A method of identifying a genomic feature present within a visiblechromosome region, comprising steps of: (a) micro-dissecting a singlecopy of a chromosome to obtain a visible chromosome region containing agenomic feature; (b) amplifying said visible chromosome region to obtainamplified single chromosome DNA; and (c) subjecting said amplifiedsingle chromosome DNA to micro-array analysis using a micro-array toidentify the genomic feature present within said visible chromosomeregion.
 2. The method according to claim 1 wherein the genomic featureis genomic DNA size, gene content, DNA breakpoint, or DNA polymorphism.3. The method according to claim 1 wherein the genomic feature is asingle nucleotide polymorphism (SNP).
 4. The method according to claim 1wherein the genomic feature comprises a plurality of single nucleotidepolymorphisms (SNPs) corresponding to a SNP haplotype of the visiblechromosome region.
 5. The method according to claim 1 wherein saidmicro-array is a genomic array, gene array, or single nucleotidepolymorphism (SNP) array.
 6. The method according to claim 1 whereinsaid micro-array is an oligo- or genomic clone-based comparative genomichybridization (CGH) array.
 7. The method according to claim 1 whereinthe visible chromosome region comprises a visible aberration.
 8. Themethod according to claim 1 wherein said chromosome micro-dissected instep (a) is obtained from a single cell isolated from a population ofheterogeneous cells.
 9. The method according to claim 1 wherein saidchromosome micro-dissected in step (a) is a human chromosome.
 10. Themethod according to claim 1 wherein said chromosome micro-dissected instep (a) is an abnormal chromosome obtained from a single dividingcancer cell.
 11. The method according to claim 1 wherein said chromosomemicro-dissected in step (a) is a normal chromosome obtained from asingle dividing cell.
 12. The method according to claim 1 wherein theamplification recited in step (b) is carried out by polymerase chainreaction (PCR), multiple displacement amplification (MDA), or acombination thereof.
 13. The method according to claim 1 including thefurther step of performing reverse fluorescence in situ hybridization(FISH) on a chromosome preparation using said amplified singlechromosome DNA to identify a genomic origin of said visible chromosomeregion, said genomic origin at least in part directing selection of anarray in the micro-array analysis of step (c).
 14. A method ofidentifying at least one single nucleotide polymorphism (SNP) presentwithin a visible chromosome region, comprising steps of: (a)micro-dissecting a single copy of a chromosome to obtain a visiblechromosome region containing a SNP; (b) amplifying said visiblechromosome region to obtain amplified single chromosome DNA; and (c)subjecting said amplified single chromosome DNA to SNP micro-arrayanalysis using a SNP micro-array to identify the SNP present within saidvisible chromosome region.
 15. The method according to claim 14 whereina plurality of single nucleotide polymorphisms (SNPs) are identified bythe method, said plurality of SNPs corresponding to a SNP haplotype ofthe visible chromosome region.
 16. The method according to claim 14wherein steps (a)-(d) are repeated using a haploid chromosome homologuesuch that SNP haplotypes of a homologue chromosome set are identified bythe method.
 17. A method of determining the identity of a singlenucleotide polymorphism (SNP) at a SNP locus present within a visiblechromosome region, comprising steps of: (a) micro-dissecting a singlecopy of a chromosome to obtain a visible chromosome region containing aSNP locus; (b) amplifying said visible chromosome region to obtainamplified single chromosome DNA; and (c) subjecting said amplifiedsingle chromosome DNA to SNP analysis to determine the identity of asingle nucleotide polymorphism (SNP) at the SNP locus present within thevisible chromosome region.
 18. The method according to claim 17 whereinthe SNP analysis of step (c) is performed by subjecting said amplifiedsingle chromosome DNA to micro-array analysis.
 19. The method accordingto claim 17 wherein step (c) is repeated to obtain a plurality of singlenucleotide polymorphisms (SNPs), said plurality of SNPs corresponding toa SNP haplotype of the visible chromosome region.
 20. The methodaccording to claim 17 wherein steps (a)-(c) are repeated using acorresponding haploid chromosome homologue such that SNP haplotypes of achromosome homologue set are identified by the method.